G protein-coupled receptors (GPCRs) play an essential role in coordinating the ability of cells to rapidly respond to their environment. Agonist binding to a GPCR promotes initial activation of heterotrimeric G proteins, which mediates downstream signaling. Agonist-occupied GPCRs then interact with GPCR kinases (GRKs), which specifically phosphorylate the receptor, and arrestins, which bind to GRK-phosphorylated receptors and function in GPCR desensitization, endocytosis, and signaling. A central question that drives current GPCR research involves understanding the dynamics and structures of GPCR interactions with G proteins, GRKs and arrestins. My laboratory is currently using a variety of strategies including X-ray crystallography, biochemical and biophysical analysis, and molecular and cellular biology to better understand the dynamics of GPCR regulation by GRKs and arrestins and the potential role of these processes in disease. In this application, we propose to address four questions that are central to understanding the mechanisms involved in GRK and arrestin regulation of GPCR signaling and trafficking. Can we obtain structural and dynamic insight on a GPCR/GRK complex? While we currently know little about the critical regions that mediate GRK interaction with GPCRs or how this interaction ultimately regulates GRK activation, our preliminary studies reveal three binding ?hot spots? between the ?2-adrenergic receptor (?2AR) and GRK5 and suggest large conformational changes in GRK5 upon binding to the receptor. How does ?-arrestin interact with the ?2-adrenergic receptor and ?2-adaptin? ?-arrestins play a central role in regulating GPCR signaling and trafficking so further understanding these interactions has important implications. Moreover, structural differences in the receptor when bound to its major downstream targets should provide insight on candidate regions to target in order to selectively enhance or disrupt specific interactions. Do ?-arrestins play a broad role in regulating GPCR trafficking and signaling? We have found that the ?-arrestin ARRDC3 functions as a switch to modulate the endosomal residence time and subsequent intracellular signaling of the ?2AR. We will perform structure/function analysis of ARRDC3 interaction with the ?2AR and assess whether ARRDCs have a broad role in regulating GPCR function. What is the molecular and structural basis of biased agonism? While it is evident that biased signaling through a number of GPCRs may ultimately prove to be of significant therapeutic value, literally nothing is known about the structural and mechanistic basis by which ligands can bias GPCR signaling. Overall, a more detailed understanding into these questions has tremendous implications for the development of more effective drugs to treat a wide variety of diseases.